1. Related Applications
The present invention contains subject matter useful in connection with the teachings of copending U.S. application Ser. Nos. 392,111 now allowed U.S. Pat. No. 4,478,946 and 399,093 claiming the priority of South African application Nos. 81/4481 and 81/4898 respectively of which the present applicant is a coapplicant. By the same token the present application discloses certain preferred embodiments which can be combined with advantage with teachings of the said copending applications.
The present invention relates to immunological preparations, their manufacture and use.
2. The Prior Art
It is known that birds, e.g. laying hens, transfer their immunity to the yolk of their eggs and thereby to their offspring (F. W. Rogers Brambell "The transmission of passive immunity from mother to young" (1970) North Holland Publishing Company, Amsterdam, London).
The present invention is based on the development of novel or improved techniques for putting this phenomenon to practical use, thereby to achieve a number of novel results and advantages.
The invention deals with a number of aspects each requiring a separate consideration of the relevant prior art:
(1) the eliciting of antibodies in fowl eggs and the cropping of the eggs for purposes of antibody recovery
(2) the recovery and purification of the antibodies from fowl eggs;
(3) novel uses of antibodies recovered from fowl eggs.
In Williams, Methods in Immunology and Immunochemistry, Ace Press NY, vol. 1, 1967 pages 209-212, 229, 337, 224-245 there is described inter alia the active immunisation of fowls, male and female, against a variety of immunogens and the recovery of antibodies from the fowl serum. However, there is no disclosure of the transfer of antibodies to the eggs of hens where such antibodies have been elicited against immunogens which are non-pathogenic in hens.
Aulesio et alia, PSEBM (1969) 131, 1150-1153 describe immunising fowl hens against microbes which are pathogenic in fowls and the resulting antibody levels were determined both in the hen serum and the egg yolk. The purpose of this work was the development of methods for monitoring chicken flocks for evidence of exposure to microbal infections. No suggestion is made to put the antibodies themselves to any practical use, nor is any method disclosed which could be put to practical use for the commercial manufacture of egg yolk antibodies. Quite clearly, the immunisation regimens employed had not yet led to optimised antibody levels in the yolk. The results strongly suggest a connection between pathogenicity of the immunogens and immunoresponse. It is stated that subsequent actual infection with pathogens (by injection of living rickettsiae) resulted in a manifold further rise of the antibody titre.
Stedman et alia. J Comp. Path., 79 (1969) 507-516) describe the recovery of anti-Newcastle disease antibodies from the egg yolk of hens which had suffered natural exposure to Newcastle disease and which had recovered from the disease. Again, the antibody levels had not reached optimum levels, because it is suggested that hyperimmunisation of the hens would have resulted in increased titres. However, no hyperimmunisation regimen is disclosed, nor an interrelationship between a hyperimmunisation regimen and an egg cropping regimen for purposes of achieving satisfactory yields of homogeneous antibodies from egg yolk. In the conclusion egg yolks are mentioned as a possible source for specific antibodies. However, no method for achieving this is disclosed and the suggestion is qualified by doubts as to the susceptibility of hens against antigens other than Newcastle disease. The recovery method for isolating or concentrating antibodies from the egg yolk results in substantial damage to the antibodies and in substantial losses. The damage is manifested in an "inherent instability of the immunoglobulins" and in substantial aggregation, the dimer content alone amounting to about 20%. Moreover, the fractionation and purification procedure involves a sequence of 8 steps (or more if the repetition of certain steps is counted as separate steps). The procedure has to be carried out at very low temperature and takes more than two days to complete.
Goudswaard et alia, Poultry Sci. vol. 56, no. 6 (November 1977), 1847-1841 describe the recovery of naturally occurring antibodies from egg white and egg yolk. The differences between fowl and mammalian antibodies are clearly not recognised. The antibodies here being dealt with are those which occur in the fowl due to natural exposure to disease. The purification method (as in Stedman et alia, see above) uses for defatting a surfactant, namely dextran sulphate--which in contrast to ordinary dextran is a potent ionic surfactant, followed by dialysis, salting out and chromatography on Sephadex and/or DEAE cellulose. Again the methods are such that they result in substantial damage to the egg antibodies.
Yamamoto et alia, Jap. J. vet. res., 23, 131-140 (1975) describe the immunisation of hens against sheep red cells by intravenous injection three times daily followed immediately by egg collection up to the 28th day from the beginning of the immunisation. Between the 10th and the 19th day of this regimen the egg yolk was found to contain small amounts of antibodies, mostly IgM (a macro-globulin which is undesirable for use in most immuno assays) and very little IgG. As from the 25th day the antibody level had dropped to negligible levels. This reference clearly contains no teaching which would encourage the use of egg yolk as a commercial source of antibodies and in fact the methods there disclosed are unsuitable for that purpose. Moreover, the minute quantities of IgG antibodies which appear in the egg yolk for a few days only, following the immunisation regimen disclosed in the reference, whilst being of scientific interest, have inferior properties for most practical purposes. The method employed for recovering and purifying the antibodies involves separating the yolk, high speed centrifugation and salting out with ammonium sulphate, i.e. methods similar to the aforementioned prior art. This recovery method, as is the case for all the prior art methods referred to above, results in substantial damage to and losses of the desired fowl egg antibodies. The extent of this damage and of these losses, as well as the avoidability thereof had not been known or appreciated according to the prior art.
The prior art did not appreciate and make allowance for the fact that there exist substantial differences between the gamma-globulins which occur in the egg yolk of fowl eggs on the one hand and mammalic (including human) gammaglobulins on the other hand. Accordingly it was not realised that due to those differences the aforementioned prior art procedures which had been applied successfully to the purification of mammalic antibodies are unsuitable for the recovery without substantial losses of undamaged, stable and non-aggregated gammaglobulins from egg yolk.
Firstly the source materials from which the gammaglobulins are recovered are substantially different. Conventional mammalic immunoglobulin preparations have mostly been recovered from blood serum. This is obtained from whole blood after removal of fibrinogen by clotting. The serum is a clear liquid containing about 6.5 to 7% by volume of dissolved proteins represented by about 0.8 to 1% gammaglobulin, 3.5% albumin, 2.5% miscellaneous proteins (including glyco-proteins, beta-lipo-proteins, IgG, ferritin, prealbumin I and II. There is no caseinaceous matter in mammalic serum and little or no free lipid matter. By way of contrast, egg yolk contains about 25% by volume yolk granules. A further approximately 25% by volume is composed of about equal parts, i.e., about 10 to 15% by volume each of caseinaceous proteins and free lipids. The remaining 50% by volume contain 5% proteins in aqueous solution (sometimes referred to as "alpha-, beta- and gamma-vitellines"). These are represented by about 2.5 to 3.6% gammaglobulin, 2.5 to 1.4% betaglobulin (in about equal parts, besides other minor contaminants. The albumins and beta-globulins of egg yolk are different from the albumins and beta-globulins in mammalian blood.
Secondly, and perhaps most important of all, the immunoglobulins themselves in egg yolk are chemically and physically significantly different from immunoglobulins in human or other mammalian serum. These differences, some of which were only discovered in the course of making the present invention, are inter alia the following:
(i) different amino acid compositions and sequences in the basic molecules,
(ii) different electrophoretic mobilities (IgY has much higher mobility than the corresponding mammalian serum IgG)
(iii) materially different isoelectric pH values, namely about 5.8 in the case of egg yolk gammaglobulin and about 6.8 in the case of mammalian serum IgG. This in turn is evidence of material difference in the ratios of carboxyl and amino groups (egg yolk gammaglobulin having relatively more of the former).
(iv) Different molecular weights, namely 175000 daltons in the case of egg yolk gammaglobulins as compared with 150000 daltons for mammalian IgG.
(v) Substantially different chemical stabilities, for which reason as stated above egg yolk gammaglobulins suffer severe damage when subjected to the conditions of most conventional purification processes which have been used successfully for IgG from mammals. Egg yolk gammaglobulin requires the presence of non-ionic surfactants as stabilisers in certain conditions where mammalian IgG is stable without such surfactant.
(vi) Ionic detergents inhibit the antigen-antibody reaction of mammalic IgG, but do not (at least not at modest concentrations) have an effect on the antibody-antigen reaction of egg yolk gammaglobulin.
(vii) Mammalic IgG remains in the monomeric form in low and high molar salt solution. IgY is monomeric in 0.15 molar NaCl and is dimeric in 1.5M NaCl.
U.S. Pat. No. 3,415,304 describes and claims inter alia processes for concentrating and purifying mammalian gammaglobulins from blood serum using water-soluble, linear, filamentary, non-charged polymers such as polyethylene glycol as a precipitant and fractionating agent. Since then a considerable number of modifications for special practical applications of that process have been published. However, in the field of isolating or purifying mammalian immunoglobulins these fractionating or purification processes have only been one type out of a large number of alternative available types, e.g. cryoethanol fractionation, precipitation with chloroform or other solvents or various salting out procedures, e.g. with ammonium sulphate. Moreover the polyalkyleneglycol methods for purifying mammalian serum gammaglobulins were designed to be applied to gammaglobulin concentrates which had already been subjected to substantial prepurification, and none of these concentrates, let alone their source materials, are at all similar in composition to fowl egg yolk.
To date no method has existed suitable for fractionating gammaglobulins of a given class (e.g. mammalian serum gammaglobulins or fowl egg yolk gammaglobulins) on a commercial scale for the purpose of concentrating or purifying antibodies with a specificity against particular antigens from antibodies against other antigens.
Immunological preparations comprising antibodies can be put to a variety of uses, including the passive immunisation of animals (including humans) and to an ever increasing extent, as immuno regants for immunosorbtive processes and in particular for quantitative and qualitative analytical tests, in particular micro assays for diagnostic, pathological, forensic and pharmacokinetic investigations.
In the therapeutic field there is a need for greater variety and specificity and improved hypo-alergenicity, bearing in mind that conventional preparations based on mammalic sera are inclined to lead to allergic reactions including anaphylactic shock.
The conventional methods of producing passive immunising preparations against specific conditions, e.g. anti-tetanus preparations, anti-venines and anti-toxins involve the immunisation of mammals such as horses, sheep, goats and the bleeding of such animals for the recovery of the antibodies from the blood. Similar procedures, sometimes also involving smaller mammals such as rabbits or guinea pigs are employed in the production of antibodies for diagnostic and similar micro-analytical testing procedures. The amounts of immunogen required for immunising a single animal are comparatively large. The immunising period is comparatively long and the bleeding of the animals is traumatic for the animals, eventually affects the health of the animals and is unpleasant for the person(s) having to recover the blood besides being often quite difficult and involving specialised skills.
It is quite generally an object of the invention to provide improvements in the aforegoing regard.
It is an object of the invention to provide fowl eggs as a practical and convenient source of a wide variety of antibodies, more particularly antibodies of a character novel per se.
It is a further object of the invention to improve the economics of antibody production by the prolonged recovery of antibody bearing eggs during the course of the laying periods of hens.
It is a further object to produce useful or increased yields of antibodies and antibodies of improved quality, elicited by immunogenic determinants, including those which in the past had modest to poor immunogenic effectiveness of hens, in particular from haptens and immunogenic determinant-bearing molecules of relatively low molecular weight, but which nevertheless produce a satisfactory immunoresponse in mammals.
It is an object to provide a method by which new antibodies or specific antibodies can be produced at short notice in a comparatively short period of time, namely a shorter period of time than with most laboratory animals conventionally used for the purpose, to provide at relatively low cost either small amounts of antibodies for specialised purposes or larger amounts and if desired continuous supplies of such antibodies over prolonged periods with minimum distress and discomfort to test animals. It is a further object to provide antibodies of particularly high selectivity and avidity.
It is a further object to provide a method suitable for eliciting antibodies in relatively large amounts, even when only very small amounts of immunogenic determinant material are available.
It is yet a further object to produce egg yolk antibodies of high homogeneity, high purity, freedom from proteolytic enzymes, high stability, being substantially undamaged and free of dimers or higher aggregates.
Further objects, advantages and uses of the invention will become apparent from what follows.